Control Reactions with ProtoScript® M-MuLV Taq RT-PCR Kit

Protocol

The following control reactions can be used to examine the quality of kit components and RT-PCR products produced by the kits. The positive control reaction should give a 327 bp fragment, and no product is detectable in the -RT Reaction (Figure 3). If a PCR product is detected in the -RT control reaction, it is due to either the contamination of genomic DNA or a carry-over PCR product.

Positive Control

-RT control

10X RT Buffer

2 μl

2 μl

Murine RNase Inhibitor

0.5 μl

0.5 μl

Rat Liver Total RNA (500 ng/μl)

1 μl

1 μl

dT23 VN (50 μM)

2 μl

2 μl

dNTP mix (2.5 mM)

4 μl

4 μl

M-MuLV Reverse Transcriptase

1 μl

-

Nuclease-free H20

9.5 μl

10.5 μl

Final volume

20 μl

20 μl

Mix well by pipetting and incubate at 42°C for one hour.

Inactivate the reverse transcriptase by heating at 80°C for 5 minutes.

Dilute the cDNA by adding 30 μl H2O, and add the diluted DNA to the following PCR reaction:

Taq 2X Master Mix

25 μl

GAPDH Primer Set (10 μM)

1 μl

Diluted cDNA

2 μl

H2O

22 μl

Total Volume

50 μl

The following PCR cycling conditions are recommended:

INITIAL DENATURATION

94°C

2 MINUTES

30 Cycles

94°C

30 seconds

55°C

15 seconds

68°C

30 seconds

Final Extension

68°C

5 minutes

Analyze 5 μl of the reaction on a 1% agarose gel, stained with ethidium bromide.